Antibody arrays are useful for detecting multiple proteins simultaneously in a biological sample. An important and growing application area for the study of multiple proteins is the identification and quantitative measurement of proteins having similar amino acid sequences, for example, protein isoforms resulting from gene-level events, for example, gene splicing and genetic translocations.
U.S. patent application publication number US 2004-0029292 A1 describes technology that can be used to identify multiple proteins using an array. Briefly, starting from the primary amino acid sequence of any protein, one can identify a series of linear epitopes (PETs) that uniquely represent the protein. By denaturing and fragmenting the protein prior to analysis, these unique regions can be exposed and separated individually or in groups. Antibodies specific to these unique regions can then be used, for example, for unambiguous protein assignment and quantitative measurement.
However, mammalian genes can undergo modifications that yield modified protein forms with similar amino acid sequences, and only limited unique regions. For example, mammalian genes are typically arranged on chromosomes in an exon-intron structure. Once the DNA is transcribed into pre-mRNA, the introns are excised in a process called splicing. Alternative splicing can occur when the introns of a pre-mRNA can be spliced in more than one way, yielding several possible mature mRNA species for a given gene. FIG. 1 is a schematic drawing that illustrates mRNA alternative splicing, which gives rise to different protein products with similar amino acid sequences when the alternatively spliced mRNAs are translated.
RNA splicing multiplies the number of potential protein biomarkers, diagnostics, and targets as compared to conventional gene arrays. There is evidence that the majority of human genes are alternately spliced, meaning that each gene may encode multiple RNA and protein products, for example, multiple splice isoforms. Splice isoforms of the same gene often have different, and even opposite, functions. Increasingly, researchers are focusing on specific splice isoforms rather than mere genes in their efforts to understand the mechanisms behind diseases.
Furthermore, there is a growing body of research that shows splice isoforms are tissue-specific, disease-specific, and/or population-specific, specific to individuals, and/or related to drug response. Therefore, alternative splicing is an important regulatory mechanism, often controlled by developmental or tissue-specific factors or even by pathological state. Through variable inclusion or exclusion of exons, it allows a single gene to generate multiple RNAs, which can be translated into functionally and structurally distinct isoforms with similar amino acid sequences.
The body's ability to generate multiple, distinct proteins with similar amino acid sequences is not unique to the phenomenon of alternative splicing. For example, in Huntington's disease, partial processing of intact disease-related protein (such as the HD protein) leads to generation of protein fragments encompassing different portions of the intact protein, or different lengths of poly-Glutamine stretch encoded by the CAG repeats. These partial proteins, in a sense, are related to one another the same way the different alternative splicing isoforms are related to one another. Thus, detection and/or quantitation of multiple, distinct proteins with similar amino acid sequences, for example, protein isoforms arising from gene-level events, can be useful for disease diagnosis.
Since one or more distinct proteins with similar amino acid sequences may be present in the same protein sample to be analyzed, discrimination of these proteins is a challenging application. For example, isoforms produced from a single gene can have large amounts of sequence identity with each other, depending, for example, upon the exons shared.
Unfortunately, the present methods for analyzing distinct proteins with similar amino acid sequences in a sample are inadequate. Many assays rely on the use of junction regions. As a skilled artisan will appreciate, alternative isoforms may contain unique junction regions, created, for example, by the fusion of different exons relative to other splicing isoforms of the same protein. However, the choice of sequences for raising antibodies that recognize the uniqueness at the junction region is limited based on the amino acids comprising the junction region, and such limited choices may not even be desirable. For example, the junction region may be too hydrophobic, too short, etc., making such regions poor candidates for raising effective capture agents (e.g., antibodies or functional fragments thereof). Consequently, it is not always possible to develop antibodies to the junction region. Further, while sequences at the junction region are generally unique relative to the isoform family, they may not be unique across the entire proteome. In fact, they may not even be unique for all the other proteins in a given sample to be analyzed.
Thus, antibodies raised to linear epitopes spanning the splice junction regions, combined with denaturing and fragmenting the sample prior to analysis, represent only a partial and limited solution for detection of distinct proteins with similar amino acid sequences, for example, protein isoforms arising from genetic events, within a protein sample. Accordingly, there is a need for a more complete and comprehensive solution to this problem.